The testis and epididymis contain many of the biochemical elements that play important roles in delivering retinoids to and within target cells for their subsequent action or metabolism. These include the extracellular retinoid transport proteins, retinol-binding protein (RBP) and lumenal epididymal retinoic acid-binding protein (E-RABP); the intracellular retinoid-binding proteins, cellular retinol-binding protein (CRABP), cellular retinoic acid-binding protein (CRABP); a novel member of the same family, testis lipid-binding protein (TLBP); the enzymes that metabolize retinoids to active and inactive forms or possible novel metabolites. Studies of the mode of delivery and metabolism of retinoids in the testis and epididymis may reveal mechanisms of regulation of production and action that may be paradigms for other sites of retinoid action in the body, as well as to contribute to our understanding of the essential roles that retinoids play in maintaining male reproductive function. The three areas of investigation involve both isolated cells and extracts from the testis and epididymis. Immunolocalization at the electron microscopic level, structural determinations by mass spectrometry, and standard biochemical and molecular biological techniques, including quantitative RT-PCR will be used. I. A model for retinoid movement/metabolism in the testis is that retinol passes through both the myoid and Sertoli cell by uptake and then release on newly synthesized RBP to reach the late germ cells and that metabolism of retinol to retinoic acid probably occurs in Sertoli cells. This will be tested by determining the uptake and metabolism of retinol from RBP by isolated cells to delineate sites of synthesis of the retinoic acid required for certain steps of spermatogenesis. A restriction of CRABP to the cytoplasm in later stage spermatogonia suggests that these cells are protected from the action of retinoic acid. Cytochrome P-450 metabolism of retinoic acid-CRABP will be determined for isolated cells and the restriction of CRABP to the cytoplasm will be studied to test this. Localization of RBP and CRABP at the EM level will test the model. II. Testicular spermatozoa accumulate retinyl ester which is depleted during epididymal transit. One hypothesis is that they metabolize the ester to retinoic acid to be released as a spermatozoa-generated signal to the epididymal cells, transported via E-RABP. This will be tested by examining spermatozoan metabolism of retinyl ester; determining if lumenal retinoids, particularly all-trans- or 9-cis-retinoic acid, shown to bind to E-RABP, will affect the expression by principal cells of specific genes known to be regulated by spermatozoa associated factor(s), particularly for those principal cells shown to contain CRABP in their stereocilia. The role of the caput principal cells that synthesize E-RABP and are rich in CRBP to this process will be examined. III. TLBP, restricted to late germ cells, will be characterized. Its endogenous ligand will be determined. Recombinant and native protein will be used for studies of binding ability and specificity.